Caenorhabditis elegans infection model for Coxiella burnetii

 描述（由申请方提供）：贝氏柯克斯体是一种极具感染性的细胞内细菌，可引起人体Q热，被归类为选择性病原体。Q热通常表现为一种使人衰弱的流感样疾病，伴有肺炎或肝炎， 但在少数情况下，严重的慢性感染会发生危及生命的心内膜炎， 主要表现。尽管这些因子和活动在病原体的生存中起着核心作用，但人们对柯克斯体的毒力决定因素或细菌如何破坏宿主细胞知之甚少。我们的总体目标是确定新的C. Burnetii的，并表征宿主在感染期间针对细菌的先天免疫应答。为此，我们最近在秀丽隐杆线虫中开发了一种新的柯克斯体感染动物模型。当C.线虫以C.伯内特氏菌，肠道成为持续殖民。此外，感染显著降低蠕虫的寿命，并产生可见和明显的病理（肛门区变形或DAR）。以往的报道表明，在C.线虫感染模型在更高级的宿主中同样被利用，并且C.线虫对病原体的先天免疫反应（IR）在高等真核生物中是保守的，强调了该模型对研究人类病原体的价值。我们假设C.线虫感染模型可以以高通量的方式用于鉴定Coxiella的新毒力因子，并分析蠕虫对这种神秘细菌的先天免疫应答的效应物。为了解决这个假设，Aim 1将筛选一个C. Burnetii Himar1 TnA7突变体在C. elegans来鉴定潜在的毒力因子。一旦确定， 相应的柯克斯体基因将使用分子科赫假设进行研究，以验证它们在增强毒力中的作用。TNSeq也将被用来分析 在感染过程中，Coxiella文库中的每个诱变基因与细菌适应性的关系。第二，我们将分析C.通过RNASeq鉴定针对秀丽隐杆线虫的先天IR效应子。伯内特氏菌在目标3中，我们将使用RNAi和C.线虫的宿主易感性调查的柯克斯体感染，相对于野生型线虫。最后，这项研究将显著推动该领域的发展：a）鉴定和分析新的柯克斯体毒力决定因子，B）提供一种新的柯克斯体动物模型。贝氏体感染，这将有助于筛选毒力因子和其他期望的表型，和c）表征潜在的新的先天IR效应物，其被宿主用来对抗病原体。

 DESCRIPTION (provided by applicant): Coxiella burnetii is an extremely infectious, intracellular bacterium that causes Q fever in humans and is classified as a select agent. Q fever typically presents as a debilitating, flu-like illness accompanied by pneumonia or hepatitis, but in a minority of cases a severe, chronic infection occurs with life-threatening endocarditis as the predominant manifestation. Little is known about Coxiella's virulence determinants or how the bacterium subverts the host cell, despite the central role for these factors and activities in the pathogen's survival. Our overall goals are to identify novel virulence determinants of C. burnetii and characterize the host's innate immune response against the bacterium during infection. To that end, we recently developed a novel animal model for Coxiella infection in Caenorhabditis elegans. When C. elegans nematodes feed on C. burnetii, the intestines become persistently colonized. Moreover, infection significantly decreases the worm's lifespan and produces a visible and pronounced pathology (deformed anal region or DAR). Previous reports showing that virulence factors identified in the C. elegans infection model are similarly utilized in higher-order hosts and that signaling pathways of C. elegans' innate immune response (IR) against pathogens are conserved in higher eukaryotes, underscores the value of this model for investigating human pathogens. We hypothesize that the C. elegans model of infection can be utilized in a high-throughput fashion to identify novel virulence factors of Coxiella and to analyze effectors of the worm's innate immune response against this enigmatic bacterium. To address the hypothesis, Aim 1 will screen a library of C. burnetii Himar1 TnA7 mutants for reduced fitness in C. elegans to identify potential virulence factors. Once identified, the corresponding Coxiella genes will be investigated using molecular Koch's postulates to verify their role in potentiating virulence. TNSeq will also be done to analyze the contribution of every mutagenized gene in the Coxiella library to bacterial fitness over the course of infection. I Aim 2, we will analyze the "infection-specific" transcriptome of C. elegans by RNASeq to identify innate IR effectors against C. burnetii. In Aim 3, we will investigate the functionality of these innate IR effectors using a combination of RNAi and mutant strains of C. elegans to investigate host susceptibility to a Coxiella infection, relative to wild-type nematodes. In the end, this stud will significantly move the field forward by: a) identification and analysis of novel Coxiella virulence determinants, b) providing a new animal model of C. burnetii infection that will facilitae screening for virulence factors and other desirable phenotypes, and c) characterizing potentially novel, innate IR effectors that are utilized by a host to counter the pathogen.